Jacqueline A. Cutler

Cellular and molecular basis of von Willebrand disease: studies on blood outgrowth endothelial cells

Von Willebrand disease (VWD) is a heterogeneous bleeding disorder caused by decrease or dysfunction of von Willebrand factor (VWF). A wide range of mutations in the VWF gene have been characterized; however, their cellular consequences are still poorly understood. Here we have used a recently developed approach to study the molecular and cellular basis of VWD. We isolated blood outgrowth endothelial cells (BOECs) from peripheral blood of 4 type 1 VWD and 4 type 2 VWD patients and 9 healthy controls. We confirmed the endothelial lineage of BOECs, then measured VWF messenger RNA (mRNA) and protein levels (before and after stimulation) and VWF multimers. Decreased mRNA levels were predictive of plasma VWF levels in type 1 VWD, confirming a defect in VWF synthesis. However, BOECs from this group of patients also showed defects in processing, storage, and/or secretion of VWF. Levels of VWF mRNA and protein were normal in BOECs from 3 type 2 VWD patients, supporting the dysfunctional VWF model. However, 1 type 2M patient showed decreased VWF synthesis and storage, indicating a complex cellular defect. These results demonstrate for the first time that isolation of endothelial cells from VWD patients provides novel insight into cellular mechanisms of the disease.

Eighteen unrelated patients with factor XI deficiency, four novel mutations and a 100% detection rate by denaturing high‐performance liquid chromatography

Summary.  Factor XI (FXI) deficiency is an autosomal bleeding disorder of variable severity. Inheritance is not completely recessive as heterozygotes may display a distinct, if mild, bleeding tendency. Eighteen unrelated FXI‐deficient patients were screened blind by fluorescent single‐stranded conformation polymorphism (F‐SSCP) analysis and denaturing high‐performance liquid chromatography (dHPLC). Mutations were detected in 14 of the 18 patients (∼78%) by F‐SSCP and in all 18 patients by dHPLC. Dideoxy sequencing confirmed the mutations in all 18 patients: eight of the mutations being novel (four of which were in previously reported patients). This showed dHPLC to be a highly sensitive, reliable technique for mutation screening in heterogeneous disorders.

Germline mosaicism resulting in the transmission of severe hemophilia B from a grandfather with a mild deficiency

We report a family in which the normal pattern of X‐linked inheritance of hemophilia B (Factor IX deficiency) is complicated by mosaicism in the probands maternal grandfather. The proband, an infant with severe Factor IX deficiency, was initially thought to be a sporadic case. Testing of other family members identified his mother as a carrier of the disorder, and his asymptomatic maternal grandfather as having very mild FIX deficiency. The causative familial mutation was identified as a two base pair deletion (AG within codons 134–135) in the Factor IX gene. The grandfather was shown to be “heterozygous” for the deletion. Karyotype analysis confirmed him to be 46XY thereby ruling out Klinefelter syndrome. The probands aunt, who as the daughter of a man with hemophilia is theoretically an obligate carrier, was found not to carry this familial mutation, and thus not to be a carrier of hemophilia B. The grandfather must therefore be an X chromosome somatic and germline mosaic, with consequent segregation of the affected and non‐affected Factor IX genes. This observation underlines the importance of confirming carrier status even in those individuals assumed to be obligate carriers, and has implications for genetic counseling.

Molecular characterization of 11 novel mutations in patients with heterozygous and homozygous FV deficiency

Summary.  Coagulation factor V (FV) has an important role in the blood coagulation cascade, in both the pro‐ and anticoagulant pathways. FV deficiency is a rare bleeding disorder with variable phenotypic expression. We report a cohort of 10 patients with mild‐severe FV deficiency in whom a total of 11 novel mutations were identified. Three patients were compound heterozygous for two mutations, whereas each of the remaining patients had a single heterozygous variant. FV levels did not correlate with either the type of mutation identified or the bleeding diathesis exhibited by the patients. Although considered to have an autosomal recessive mode of inheritance, patients with a single missense mutation may present with a significant bleeding history. The addition of a significant number of previously unidentified mutations to the public domain will contribute to the knowledge and understanding of the molecular pathology of this rare disorder.

Characterization of a causative mutation of hemophilia A identified in the promoter region of the factor VIII gene (F8)

Hemophilia A is an X-linked bleeding disorder of variable severity resulting from deficient or dysfunctional factor VIII protein. Genetic defects of the F8 gene are heterogeneous [1], and all the mutations reported in the Haemophilia A mutation Database [2] affect or disrupt theF8 gene coding sequence or its intron–exon boundaries. However, in 1 to 2% of cases, a causative mutation cannot be identified in the F8 gene [3], and prior to the recent publication by Bogdanova et al. [4], no causative mutations had been described in the F8 gene promoter. The F8 gene is regulated by a 1175-bp promoter that contains at least 19 binding sites for liver-specific and ubiquitous transcription factors, including, c/EBPa, c/EBPb, nuclear factor kappaB, HNF1, NF-Y andHLF [5–7].We have identified the same C>T substitution at position –219 (standard Human Genome Variation Society (HGVS) nomenclature; +1 refers to the first base of the ATG translational initiation codon, as per Figueiredo et al.) of the F8 gene promoter reported byBogdanova et al. [4] in a three-generation family with mild hemophilia A, and demonstrated its causality. A 61-year-old man admitted to hospital following an assault was found to have a low FVIII:C of 30.3 IU dL (normal range: 73–200 IU dL). A diagnosis of hemophilia A had not been previously assigned, but there was a strong personal history of bleeding. Molecular analysis was undertaken to determine the causative mutation of the apparent mild hemophilia A. Mutation screening, by denaturing high-performance liquid chromatography analysis, followed by dideoxy sequencing of all 26 exons and associated splice junctions of the F8 gene, failed to detect any variation from the normal sequence. Further phenotypic testing excluded an obvious alternative diagnosis, such as combined FV and FVIII deficiency or type 2N von Willebrand disease, with normal FV levels and vonWillebrand factor parameters, and a normal F8 binding assay. Subsequent analysis of the F8 gene promoter region, by polymerase chain reaction (PCR) amplification and dideoxy sequencing, detected a C>T substitution at position – 219. This base change was excluded as a common polymorphism by the failure to detect it in > 100 normal alleles. The possible influence of the –219C>T substitution on the F8 promoter was investigated. A 317-bp fragment from the human F8 gene promoter (positions –314 to +3; +1 refers to the first base of the ATG translational initiation codon) was amplified by PCR using patients or control DNA samples as a template, and ligated into a promoterless pGL3Basic luciferase reporter plasmid (Promega, Southampton, UK). PLC/PRF/5 cells (ATCC, CRL-8024), a hepatoma cell line proven to express FVIII [7,8], were cultured and transfected with both the firefly luciferase reporter plasmid (2 lg), containing either wild-type or mutant sequence, and an internal control plasmid (0.05 lg) containing the Renilla luciferase gene. After 48 h, cell lysates were assayed for luciferase activity using a dual-luciferase assay system (Promega). The firefly luciferase activity was calculated as fold induction compared to the transfected promoterless reporter plasmid, and normalized with respect to transfection efficiency using the Renilla luciferase activity. The relative value of luciferase activity for the mutant sequence was expressed as a percentage of the activity observed for the wild-type sequence. From three experiments, each with triplicate dishes, it was found that the plasmid containing the mutant sequence expressed 23% ± 3.2% (mean ± SEM) of the luciferase activity of the wild-type sequence. The results indicated that the presence of the –219T mutation significantly reduced the F8 promoter activity. Further analysis by electrophoretic mobility shift assay (EMSA) was performed to investigate the interaction of the F8 sequence with nuclear extracts from PLC/PRF/5 cells. Oligonucleotides corresponding to the wild-type and mutant sequences (positions –228 to –208 of the F8 promoter) were synthesized and then labeled with biotin-11-UTP, using a biotin 3¢-endDNA-labeling kit (Pierce, Northumberland,UK). The labeled oligonucleotides were then bound to PLC/PRF/5 cell nuclear extracts, prepared according to the method of Hoppe-Seyler et al. [9], and the protein–DNA complexes were electrophoresed on a 6% native polyacrylamide gel and Correspondence: Mike Mitchell, Centre for Haemostasis and Thrombosis, The Haemophilia Reference Centre, 1st Floor North Wing, St Thomas Hospital, London SE1 7EH, UK. Tel.: +44 0 207 188 2798; fax: +44 0 207 62

More on: unusual expression of the F9 gene in peripheral lymphocytes hinders investigation of F9 mRNA in hemophilia B patients

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The significance of published polymorphisms in 14 cases of mild factor VII deficiency

Factor VII (FVII) plays a critical role in the initiation of blood coagulation, and patients with dysfunctional or reduced levels of this protein are susceptible to mucosal bleeding. There is poor correlation between the clinical presentation and the phenotypic data; and in cases of a mild bleeding tendency, mild to moderate reductions in both FVII antigen and activity may be overlooked. The prevalence of FVII deficiency may therefore be underestimated. Polymorphic differences throughout the FVII gene are associated with variations in plasma FVII antigen and activity levels. This study highlights the significance of mild FVII deficiency, and examines the importance of seven previously published polymorphisms in such patients.

A rapid and cost-effective method for analysis of three common genetic risk factors for thrombosis.

A simple, rapid and cost-effective method for the analysis of three of the most widely screened genetic risk factors for thrombosis has been established. The protocol developed uses blood spots stored on filter paper (Guthrie spots) as well as DNA extracted from anticoagulated blood. The use of Guthrie spots taken at birth enables the retrospective study of patients who develop thrombotic complications without necessitating resampling. Following isolation of DNA, conventional fluorescence-labelled polymerase chain reaction (PCR) is performed using a thermostable DNA polymerase. Denatured, single-stranded PCR products are analysed on a semi-automated capillary-based genetic analyser, the data being stored electronically. This sensitive protocol obviates the need for endonuclease digestion and the associated gel running and documentation, and leads to a reduction in the recurrent costs of laboratory consumables.

The utility of a fast turnaround ADAMTS13 activity in the diagnosis and exclusion of thrombotic thrombocytopenic purpura

Additional Supporting Information may be found in the online version of this article: Table SI. Demographics and clinical characteristics of study population. Table SII. Cases treated with one or more platelet transfusions. Fig S1. Flowchart and description of study population. *Excluded cases did not differ from the included cases for HPA-type or, when known, the severity of the disease. Fig S2. Individual first and lowest platelet counts per postnatal treatment strategy. (A) First platelet count. (B) Lowest platelet count.

The first report of a multi-exon duplication in the F9 gene causative of severe haemophilia B

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